Previous research indicates that prisoners in Iran are at risk of drug-related harm, including acquisition of blood-borne infections. In response, several prevention interventions have been introduced into prisons in Iran, such as methadone maintenance treatment (MMT). MMT is now provided to opioid-dependent prisoners in 142 of the 230 prisons and correctional settings in Iran. A baseline behavioral survey was conducted in Karaj Central prison which mainly holds prisoners with drug-related charges. Overall, 203 male prisoners from randomly selected rooms in two prison blocks were interviewed using a structured questionnaire in 2007, just before the introduction of MMT program in this prison. Among participants, 7% reported never having used illicit drugs in their lifetime, but 51% had used non-injecting illicit drugs, and as high as 42% reported having injected an illicit drug. Up to 79% (160/203) of all participants reported using drugs, and about 6% (12/203) reported drug injecting during their current incarceration term. Same-gender sexual practice during current incarceration term was reported by 2.5% (5/203) of all male prisoners. Comparison between injecting and non-injecting drug-using prisoners indicated that drug injectors had higher rates of previous incarcerations, commenced drug use at a younger age, were more likely to have used illicit drugs in the previous week, were more likely to have been treated by a physician for drug addiction, had higher rates of registration for methadone treatment inside prison, and were more likely to have been tested for HIV infection. These study findings provide a behavioral profile of prisoners in regard to drug-related harm and can be considered in any plan to introduce or improve provision of MMT in prisons in Iran or other countries with similar features.